Peer-reviewed by 2 reviewers with median rating of 13/20. Review process was triple-blinded.

Round 1 (11/20)

Round 2 (13/20)

Technical quality4

Conceptual advance and Impact6

General comment

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The authors showed that in murine acute brain slices the internalization of two SRs ligands was very high in NeuN-positive cells and they concluded that, in cerebral cortex, neurons may have higher number of functional SRs on the surface than other cell-types. However important validations (such as colocalization with markers for other cell types, such as GFAP and Iba1) are still missing. Such validations would be important to support the authors' major conclusions.

Abstract

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When referring to the Allen's Brain Atlas, the authors should state the name of the gene investigated that show neuronal-like expression.

Figure

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See comments in 'Results & Discussion'

Objective

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In order "to investigate which brain cell type exhibited highest affinity for SRs ligands" the authors should include more than one cell type marker.

Results & Discussion

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The authors showed that in murine acute brain slices the internalization of two SRs ligands was very high in NeuN-positive cells and they concluded that cortical neurons express large amounts of functional SRs.
They claim that they detected very few non-neuronal cells internalizing those ligands, so it would be very useful to include those data.
In the figure, in fact, no other marker is shown (es. for astrocytes / for microglia) but NeuN.
- Also, could the authors provide higher magnification images for the NeuN staining?
- Could possibly provide a nuclear marker, such as DAPI?
It is important for the authors to include in the figure the colocalization of MBSA and polyG with Iba1 and GFAP, respectively.
This will be necessary to support their conclusion that "in cerebral cortex neurons may have higher number of functional SRs on the surface than other cell-types".
Importantly, which structure of the brain slice has been imaged?
In addition, the authors could also consider to perform in primary cortical neurons the same in vitro MBSA internalization assay that they have performed in Raw264.7 cells (FigA-B).

Conclusions

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As stated before,
It is important for the authors to include in the figure the colocalization of MBSA and polyG with Iba1 and GFAP, respectively.
This will be necessary to support their conclusion that "in cerebral cortex neurons may have higher number of functional SRs on the surface than other cell-types".
Showing only the colocalization with NeuN would not be sufficient.

Conjectures

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What do the authors mean for 'gene dow-regulation experiments' for assessing the question no.1 ?
The authors could also consider to assess the transcriptional profile of SRs in neurons, along with IHC.

Technical quality5

Conceptual advance and Impact6.5

General comment

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This manuscript reports a novel observation that could generate much interest. There are some technical limitations to the data that needs to be addressed in future studies through the use of inhibitors. The initial observation is appropriate as a short report for Matters. However, the overinterpretation of the data is the biggest issue with this submission.
There are no obvious data in the submission to justify the words "specifically" and "rapidly" in the title. Only a neuronal marker was used in the study and neither the cell-type selectivity nor the receptor selectivity was explored. The uptake experiment is not a kinetic study, hence the issue with the word "rapidly".

Title

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There are no obvious data in the submission to justify the words "specifically" and "rapidly" in the title.

Figure

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Not all the scale bars are 100 micrometers. The authors need to add the information for the panels A-C.

Objective

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The study did not attempt to identify all cell types that internalized the ligand. Thus, it is better to rephrase "which brain cell type" to reflect what was actually studied - uptake by neurons. The term "highest affinity" is mistakenly used since no experiment attempted to quantify the ligand binding per se.

Results & Discussion

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There is no data to support the claim "the cells which maximally endocytosed..." no quantification was performed. It is best to state that neurons robustly internalized the ligands.
The sentence "Thus, contrary to expectations, neurons rather than microglia or astrocytes have higher amounts of functional SRs in cortex." is not supported by staining of microglia or astrocytes. The authors need to reconcile. It is best to refrain from making strong statements if the data is not shown.

Conclusions

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The authors need to qualify what experiment led them to conclude "we detected very few non-neuronal cells.." what markers were used to identify non-neuronal cells. If there is some data, they can state the % of cells in the text to support this claim. If the data is not available, they need to reword this sentence based on the data at hand.

Conjectures

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It would be also important to consider that the uptake has not been convincingly demonstrated to be through SR-mediated endocytosis in this study by the use of either SR-selective inhibitors or a general clathrin-mediated endocytosis inhibitor.